FRI0271 Dimethyl fumarate inhibits ube2l3 mediated tlr7 signalling and autoreactive b cell development in sle

Background Genetic studies have identified a single UBE2L3 risk haplotype which is associated with SLE and multiple autoimmune diseases, and leads to increased expression of UBE2L3 in eQTL studies. The E2 ubiquitin-conjugating enzyme UBE2L3 regulates NF-κB activation through regulation of the Linear Ubiquitination Chain Assembly Complex (LUBAC). Thus UBE2L3 regulates CD40-driven B cell activation. The UBE2L3 risk allele correlates with circulating plasmablast and plasma cell expansion in SLE individuals. Objectives To determine the effect of UBE2L3 and linear ubiquitination on TLR7 signalling, and test the effect of Dimethyl Fumarate (DMF), which has recently been shown to inhibit UBE2L3, on B cell and plasmablast differentiation in SLE. Methods Confocal microscopy, immunoprecipitation and western blot were used to assess linear ubiquitin chain accumulation in TLR7-HEK293 cells after TLR stimulation by Resiquimod. The effect of UBE2L3 and LUBAC on TLR7 signalling was dissected by the use pf UBE2L3/LUBAC overexpression, dominant negative mutants and shRNA silencing, measuring NF-kB reporter activity, IkappaBalpha phosphorylation, gene expression by qPCR and IL-8 secretion. DMF was administered in vitro to human B cells isolated from SLE patients (n=15) and controls and cultured with Resiquimod and/or IFNα for 5–7 days. B cell viability/proliferation, plasmablast differentiation were analysed by 10-colour flow cytometry. Supernatants were assayed for immunoglobulin secretion and autoantibody production. Results TLR7 stimulation led to intracellular accumulation of linear ubiquitin chain comparable to TNFα. UBE2L3 and LUBAC co-overexpression enhanced TLR7 driven NF-kB activation and led to increased NF-κB target mRNA expression and increased secretion of IL-8. The effect was specific to UBE2L3 compared to other E2 enzymes. Dominant negative mutant UBE2L3(C86S) or HOIP(C885S) or UBE2L3/HOIP shRNA suppressed the response to TLR7 stimulation. DMF showed a dose-dependent inhibition of TLR7-mediated NF-kB activation. In primary SLE and healthy B cells, DMF suppressed proliferation of switched and unswitched CD27 +memory B cells and blocked plasmablast differentiation. DMF profoundly inhibited immunoglobulin secretion and anti-nuclear autoantibodies production in response to TLR7 and IFNa stimulus. Conclusions Our data demonstrate that linear ubiquitination and UBE2L3 regulate TLR7 activation of NF-κB. UBE2L3 silencing or pharmacological antagonism of UBE2L3 by DMF suppressed the response to TLR7 activation. Excessive TLR7 signalling has been linked to SLE development and enhanced B cell autoreactivity. Thus our data identify a novel mechanism by which the UBE2L3 risk haplotype contributes to SLE susceptibility. DMF suppressed plasmablast differentiation and inhibited TLR7 and IFNa induced autoantibody production. These results support a role for repositioning DMF (currently used to treat multiple sclerosis) in the treatment of SLE. Disclosure of Interest None declared